This proposal brings together a small focused group of four researchers to investigate fundamental biological questions associated with trinucleotide repeats (TRs). TRs are the most common type of simple sequence repeats found in the exome of all known eukaryotic genomes. They are associated with high mutation rates of the repeat number, which leads to frequent polymorphism in the coding regions of the genes with a rapid expansion of the amino acid repeats. TRs display dynamic mutations that do not follow Mendelian inheritance. One e?ect of this results is to give rise to a series of genetic diseases for which the age of disease onset and its severity increases with successive generations. The mutations responsible for these diseases are associated with intergenerational expansion of the TR. Once the repeated length of the TR reaches a certain threshold, the probability of further expansion and severity of the disease increases with the repeat length. TRs in human genes can cause severe neurodegenerative and neuromuscular disorders that lead to cell toxicity and ultimately death. While the mechanisms underlying these diseases are complex, some fundamental aspects have been recognized, such as the correlation between the repeat length and probability of further expansion and the increased pathological severity. It as also been recognized that the critical step in these diseases involves the transient formation of atypical non-B DNA stable secondary structures in the expandable repeats. The goal of this proposal is therefore to address the characterization of these atypical structures from three di?erent angles. Speci?cally, we will use large-scale atomistic simulations to address issues with regards to the conformations, relative stability, dynamics, and repeat-length dependence of atypical TR secondary structures such as slip-stranded duplexes, single-stranded hairpins, Z-DNA, DNA triplexes, etc. Second, single molecule ?uorescence resonance energy transfer (smFRET) experiments will be used to characterize the kinetics and free energies of DNA hairpin transitions. This will provide important experimental input and validation for the computational e?orts in elucidating the properties of atypical TR structures. Finally, we will use statistical genetics approaches to incorporate the role of TRs in genome evolution. By coupling data on genetic variation to our molecular dynamic simulations, we will characterize the relationship between evolutionary rates and propensity of the TRs to form atypical secondary structures. We will investigate the origin of genetic variation (mutation), intraspeci?c genetic variation (polymorphism), and inter- speci?c genetic divergence. Illuminating the link between atypical TR structures and parent-o?spring mutation data will be a much needed preliminary step toward a mechanistic understanding of the TR mutation process.